The present invention relates to a reference blood filter paper for diagnosis of homocystinuria, one kind of congenital disorder of amino acid metabolism, and more particularly to a reference blood filter paper of the kind mentioned above, which is used for the diagnosis of homocystinuria by measuring the concentration of methionine in the blood, and which can be preserved for a long period of time without any detectable deterioration.
Congenital disorders of amino acid metabolism, such as phenylketonuria, histidinemia and homocystinuria, are fearful diseases leading to mental deficiency or hepatocirrhosis. These days, however, patients with such congenital disorders can be cured and their lives saved if the disorders are identified during early infancy and the patients are promptly and appropriately treated, for instance, by subjecting them to dietary treatment.
In order to identify those disorders, it is necessary that the concentration of a particular amino acid in the blood of a new born baby be measured within a week after its birth, to determine whether or not that concentration is abnormally high. For this purpose, there is desired a simple and reliable screening assay.
The assay which is in most general use for this determination of concentration at preset is Guthrie's Bacterial Inhibition Assay. The principle of that assay is as follows:
When bacillus subtilis is cultured in an agar culture medium, if the agar culture medium contains a predetermined amount of a metabolism inhibitor which works on an amino acid which is indispensable to the growth of bacillus subtilis, the growth of bacillus subtilis will be inhibited by the action of the metabolism inhibitor. However, when a piece of filter paper into which sample blood has been infiltrated is placed on the above-mentioned agar culture medium containing the metabolism inhibitor, and bacillus subtilis is cultured there, bacillus subtilis can grow, utilizing the amino acid contained in the blood in the filter paper, so that a growth circle of bacillus subtilis, corresponding in size to the quantity of the amino acid contained in the blood, is formed.
Likewise, bacillus subtilis is cultured on a piece of reference blood filter paper into which blood containing a known amount of the amino acid has been infiltrated, so that a reference growth circle of bacillus subtilis is obtained.
By comparing the first mentioned growth circle with the second mentioned reference growth circle, the approximate concentration of the amino acid in the sample blood can be determined.
The details of this procedure and measurement conditions for the Guthrie's Bacterial Inhibition Assay are described in Rinshobyori (Clinical Pathology) 24 (12) 962-973, 1976.
In order to obtain highly stable and reproducible measurement results, it is indispensable that the amount of the amino acid contained in the reference blood filter paper not change with time, and the reference blood filter paper be preservable for a long period of time without any detectable deterioration.
In a conventional reference blood filter for measuring the concentration of methionine in the blood, methionine contained in the reference blood filter is oxidized extremely easily during preservation and, accordingly, its properties also change during preservation.
For instance, when it is preserved at a temperature of -20.degree. C., it is so deteriorated after 4 months that it cannot be used any longer. This is a significant shortcoming of the conventional reference blood filter paper.